The Wnt signaling pathway's association with carcinogenesis began as a result of early observations and experiments in certain murine mammary tumors. Wnt-1 proto-oncogene (Int-1) was originally identified from mammary tumors induced by mouse mammary tumor virus (MMTV) due to an insertion of a viral DNA sequence. Nusse et al., Cell 1982; 31: 99-109. The result of such viral integration was unregulated expression of Int-1 resulting in the formation of tumors. Vanooyen, A. et al., Cell 1984; 39: 233-240; Nusse, R. et al., Nature 1984; 307: 131-136; Tsukamoto et al., Cell 1988; 55: 619-625. Subsequent sequence analysis demonstrated that the Int-1 was a mammalian homolog of the Drosophila gene Wingless (Wg), which was implicated in development, and the terms were then combined to create “Wnt” to identify this family of proteins.
The human Wnt gene family of secreted ligands has now grown to at least 19 members (e.g., Wnt-1 (RefSeq.: NM—005430), Wnt-2 (RefSeq.: NM—003391), Wnt-2B (Wnt-13) (RefSeq.: NM—004185), Wnt-3 (ReSeq.: NM—030753), Wnt3a (RefSeq.: NM—033131), Wnt-4 (RefSeq.: NM—030761), Wnt-5A (RefSeq.: NM—003392), Wnt-5B (RefSeq.: NM—032642), Wnt-6 (RefSeq.: NM—006522), Wnt-7A (RefSeq.: NM—004625), Wnt-7B (RefSeq.: NM—058238), Wnt-8A (RefSeq.: NM—058244), Wnt-8B (RefSeq.: NM—003393), Wnt-9A (Wnt-14) (RefSeq.: NM—003395), Wnt-9B (Wnt-15) (RefSeq.: NM—003396), Wnt-10A (RefSeq.: NM—025216), Wnt-10B (RefSeq.: NM—003394), Wnt-11 (RefSeq.: NM—004626), Wnt-16 (RefSeq.: NM—016087)). Each member has varying degrees of sequence identity but all contain 23-24 conserved cysteine residues which show highly conserved spacing. McMahon, A P et al., Trends Genet. 1992; 8: 236-242; Miller, J R. Genome Biol. 2002; 3(1): 3001.1-3001.15. The Wnt proteins are small (i.e., 39-46 kD) acylated, secreted glycoproteins which play key roles in both embryogenesis and mature tissues. During embryological development, the expression of Wnt proteins is important in patterning through control of cell proliferation and determination of stem cell fate. The Wnt molecules are also palmitoylated, and thus are more hydrophobic than would be otherwise predicted by analysis of the amino acid sequence alone. Willert, K. et al, Nature 2003; 423: 448-52. The site or sites of palmitoylation are also believed to be essential for function.
The Wnt proteins act as ligands to activate the Frizzled (Frz) family of seven-pass transmembrane receptors. Ingham, P. W. Trends Genet. 1996; 12: 382-384; YangSnyder, J. et al., Curr. Biol. 1996; 6: 1302-1306; Bhanot, P. et al., Nature 1996; 382: 225-230. There are ten known members of the Frz family (e.g., Frz1, Frz2, Frz3 . . . . Frz10), each characterized by the presence of a cysteine rich domain (CRD). Huang et al., Genome Biol. 2004; 5: 234.1-234.8. There is a great degree of promiscuity between the various Wnt-Frizzled interactions, but Wnt-Frz binding must also incorporate the LDL receptor related proteins (LRP5 or LRP6) and the membrane and the cytoplasmic protein Dishevelled (Dsh) to form an active signaling complex.
The binding of Wnt to Frizzled can activate signaling via either the canonical Wnt signaling pathway, thereby resulting in stabilization and increased transcriptional activity of β-catenin [Peifer, M. et al., Development 1994; 120: 369-380; Papkoff, J. et al, Mol. Cell Biol. 1996; 16: 2128-2134] or non-canonical signaling, such as through the Wnt/planar cell polarity (Wnt/PCP) or Wnt-calcium (Wnt/Ca2+) pathway. Veeman, M. T. et al., Dev. Cell 2003; 5: 367-377.
The canonical Wnt signaling pathway is the most relevant of the Wnt signaling pathways to the development of cancer. Ilyas, M. J. Pathol. 2005; 205: 130-144. Normal activation of this pathway begins a series of downstream events culminating in the stabilization and increased levels of the protein β-catenin. This protein is normally an inactive cytoplasmic protein, and is found at the cell membrane bound to proteins including e-cadherin. In the absence of Wnt ligand, phosphorylated cytoplasmic β-catenin is normally rapidly degraded. Upon activation of the canonical pathway, unphosphorylated β-catenin is transported to the nucleus where it further results in transcriptional activation of various target genes. The subsequent upregulation in transcription of these target genes leads to changes in the cell, and continuous, unregulated expression of such target genes results in tumor development. Since aberrant Wnt signaling appears to be a necessary precursor in carcinogenesis, effective inhibitors of Wnt signaling are of great interest as cancer therapeutics.
The use of soluble receptors as antagonists to ligand-receptor interactions is known in the art. Such molecules can be effective therapeutic antagonists if they bind the free ligand in a manner so as to prevent the initial receptor activation step of the signaling pathway. Soluble minimal extracellular domain (ECD) fragments of the cysteine-rich domain (CRD) of a Frizzled receptor which exhibit binding to Wnt have been identified, based on crystallography data. Dann et al., Nature 412: 86-90 (2001). However, while such Frizzled fragments did exhibit binding to Wnt ligand, such fragments are unsuitable for therapeutics because of their rapid degradation in vivo.
The use of a soluble Frizzled domain coupled to an immunoglobulin Fc as a potential Wnt antagonist has been proposed. Therapeutic Opportunities of the Wnt Signaling Pathway in Cancer, New York Academy of Sciences, Oct. 25, 2005; Hsieh, J-C. et al., PNAS, 96: 3546-3551 (1999). However, prior to the present invention, attempts at creating a soluble Frizzled receptor-Fc fusion therapeutic were not successful. For example, one such chimera based on residues 1-173 of the Frz8 CRD (Frz (173)-Fc, SEQ ID NO: 113) had suboptimal efficacy (FIG. 12), and was unstable in vivo (FIG. 11). Moreover, the Frz (173)-Fc chimera only reduced the rate of increase in tumor volume (as opposed to shrinking starting tumor volume). Additionally, while the creation of Fc fusions is generally known as one technique to improve the in vivo stability of the resulting construct, the creation of effective therapeutic Fc constructs can be difficult owing to a number of problems, including improper protein folding of the new protein construct and steric hindrance of the fusion construct to the target.
Thus, a need exits for a Wnt antagonist therapeutic with enhanced in vivo stability that acts to inhibit Wnt ligand induced cellular signaling.